Association between Altered Expression and Genetic Variations of Transforming Growth Factor β-Smad Pathway with Chronic Myeloid Leukemia

Background: Chronic myeloid leukemia (CML) is a hematological disorder caused by fusion of BCR and ABL genes. BCR-ABL dependent and independent pathways play equally important role in CML. TGFβ-Smad pathway, an important BCR -ABL independent pathway, has scarce data in CML. Present study investigate the association between TGFβ-Smad pathway and CML. Materials and Methods: Sixty-four CML patients and age matched healthy controls (n=63) were enrolled in this study. Patients were segregated into responder and resistant groups depending on their response to Imatinib mesylate (IM). TGFβ1 serum levels were evaluated by ELISA and transcript levels of TGFβ1 receptors, SMAD4 and SMAD7 were evaluated by Real-Time PCR. Sequencing of exons and exon-intron boundaries of study genes was performed using Next Generation Sequencing (NGS) in 20 CML patients. Statistical analysis was performed using SPSS version 16.0. Results: TGFβ1 serum levels were significantly elevated (p = 0.02) and TGFβR2 and SMAD4 were significantly down-regulated (p = 0.012 and p = 0.043 respectively) in the patients. c.69A>G in TGFβ1, c.1024+24G>A in TGFβR1 and g.46474746C>T in SMAD7 were the most important genetic variants observed with their presence in 10/20, 8/20 and 7/20 patients respectively. In addition, TGFβR1 transcript levels were reduced in CML patients with c.69A>G mutation. None of the genes differed significantly in terms of expression or genetic variants between responder and resistant patient groups. Conclusion: Our findings demonstrate the role of differential expression and genetic variants of TGFβ-Smad pathway in CML. Decreased TGFβR2 and SMAD4 levels observed in the present study may be responsible for reduced tumor suppressive effects of this pathway in CML.


INTRODUCTION
Chronic Myeloid Leukemia (CML) is a hematological disorder, caused due to the transformation of pluripotent stem cells from progenitors to malignant cells. The disease process of CML initiates with the formation of Philadelphia chromosome,a unique chromosome created by reciprocal chromosomal translocation of BCR gene from chromosome 9 to chromosome 22 1 . Expression of this fusion gene is responsible for molecular alterations which increases malignant myelopoiesis and alter normal blood cell production 2 .Imatinibmesylate (IM) is the frontline therapy for CML. It is a selective tyrosine kinase inhibitor (TKI) targeted against BCR-ABL, which successfully halts disease progression towards blast phase in most of the patients 3 . However, a significant proportion of patients (approximately 20%-25%) show primary or secondary resistance to IM therapy. Hence,it is important to assess the role of other potential therapeutic targets in CML. Several molecular pathways, including BCR -ABL dependent and independent, are under investigation for their role in IM resistance 4,5 . Transforming growth factorβ (TGFβ) -Smad pathway is a multifunctional molecular pathway, which regulates different cellular activities like apoptosis, metamorphosis, differentiation, proliferation, angiogenesis, remodeling of extra-cellular matrix, etc 6 . TGFβ1, a pleiotropic cytokine, binds to receptor TGFβR2, which in turn recruits TGFβR1 7 . Activated TGFβR2/TGFβR1 complex phosphorylates the receptor-Smads (R-Smads), Smad2 and Smad3. R-Smads phosphorylate and form a higher order complex with common-Smad (Co-Smad), SMAD4, and translocates to the nucleus for expression of target genes. Inhibitory Smads (I-Smads), Smad6 and Smad7, prevent phosphorylation of R-Smads by TGFβR1. Smad6 is known to play its role in BMP-Smad pathway, whereas SMAD7 is involved in TGFβ-Smad pathway 8,9,10 . Several evidence demonstrate association of TGFβ-Smad pathway with cancer 11 . Loss of functional mutations in TGFβR2 or decreased expression of the receptor due to epigenetic changes have been reported in various cancers, including colorectal, gastric, ampullary carcinomas, gliomas, etc 12,13,14 . Further, in human oral carcinoma, metastatic cells show significantly reduced TGFβR2 levels as compared to primary tumors 15 . Low expression of SMAD4 has been reported in various cancers and is linked with better prognosis 16,17 . In hematological malignancies including AML and T-cell lymphomas, low levels of SMAD4 have been documented 18,19 . Association of TGFβ-Smad pathway with CML is not well established and needs to be explored. This encouraged us to study the expression and genetic variant in genes of this important pathway in CML. We examined the serum levels of ligand TGFβ1 and expression of its receptors TGFβR1 and TGFβR2, Co-Smad (SMAD4) and I-Smad (SMAD7) in CML patients, Association between genetic mutations in study genes and CML was also analyzed in a subset of CML patients.

MATERIALS AND METHODS Subjects
All patients (>18 years) diagnosed with CML were prospectively enrolled for a period of two years (October 2013-October 2015) at Department of Medical Oncology, Sir Ganga Ram Hospital, Delhi, India. Diagnosis was confirmed by reverse transcription polymerase chain reaction (RT-PCR) for BCR-ABL fusion gene and fluorescent in situ hybridization (FISH) for translocation (9; 22). Enrolled patients were segregated as responders and resistant as per European Leukemia Net, 2013 (ELN, 2013) recommendations. At the time of enrollment, patients' clinical and demographic data were obtained (Table 1). Healthy subjects with no known history of malignancy and above 18 years of age were enrolled as age-matched controls. The study was reviewed and approved by the Ethics Committee of Sir Ganga Ram Hospital (EC No.: EC/11/12/439). Informed consent was signed and submitted by all subjects at the time of enrollment. Peripheral blood sample in EDTA vials and plain vials (for serum) was obtained from both patients and controls. Serum was collected to compare TGFβ1 levels and stored at -80 o C. Peripheral blood RNA and DNA were immediately extracted (Nucleospin RNA#740200 and Nucleospin DNA#740951 Macheley-Nager, Duren, Germany) and stored at -80 o C for further use. Briefly, 25 ng of cDNA was used to prepare 10 µl of reaction containing respective primers and SYBRgreen. ACTB (β-actin) was used as an endogenous gene. The raw data was analyzed manually by 2 -∆Ct method and the median of 2 -∆Ct was compared between patients and controls.

Identification of genetic variants
Next Generation Sequencing A targeted panel with probes covering all coding exons and essential splice sites for TGFβ1, TGFβR1, TGFβR2, SMAD4 and SMAD7 genes was used for sequencing these samples using Illumina'sTruSight technology (Illumina, San Diego, USA). Genomic DNA isolated from blood was quantified using Qubit (Thermo Fisher Scientific, Waltham, USA) and 50 ng was taken for library preparation. Briefly, DNA was subjected to fragmentation and tagged with adaptors, platform-specific tags and barcodes to prepare the DNA sequencing libraries. The tagged and amplified sample libraries were checked for quality using BioAnalyzer (Agilent2100, Santaclaraca, USA) and quantified using Qubit. 500 ng of each library was pooled with other samples and hybridized to biotinylated probes. The hybridized target DNA fragments were pulled down using streptavidin beads. Two successive enrichment steps were performed to optimize the pull down of the regions of interest. Target libraries were amplified using limited PCR steps and loaded for sequencing on the MiSeq (Illumina, San Diego, USA) to obtain ~3 GB per sample.

Sequence analysis
The trimmed FASTQ files were generated using MiSeq Reporter from Illumina. The reads were aligned against the whole genome build hg19 using STRAND® NGS V2.1.6 (Strand Life Sciences Pvt. Ltd., Bangalore, India). Five base-pairs from the 3' end of the reads were trimmed, as were 3' end bases with quality below 10. Reads which had length less than 25 bp after trimming were not considered for alignment. A maximum of 5 matches of alignment score at least 90% were computed. Reads that failed quality control, reads with average quality less than 20, reads with ambiguous characters were all filtered out. The STRAND® NGS variant caller was used to detect variants at locations in the target regions covered by a minimum of 10 reads with at least 2 variant reads. Variants with a decibel score of at least 50 were reported. Interpretation Interpretation of the variant data was done using the Strand Omics software, V1.9. Strand Omics is a clinical genomics interpretation and reporting platform developed at Strand Life Sciences, Bangalore. The variant annotation engine includes algorithms to identify variant impact on gene using both public content (HGMD, ClinVar, OMIM, HPO, links to dbSNP, 1000 Genomes, Exome Variant Server, and built-in algorithms SIFT, PolyPhen HVAR/HDIV, Mutation Taster and LRT) and proprietary content (curated variant records). Interpretation interface in Strand Omics allows quick filtering and evaluation of variants along with capture of justification for inclusion/ exclusion. Mann-Whitney U test was used respectively. Pvalue <0.05 was considered as significant.

RESULTS
CML patients (n=64) and age-matched healthy controls (n=63) were enrolled in the study. In our patient cohort, 47 patients were IM responder and 17 were IM resistant. Demographic and clinical features of subjects are described in Table 1.  (Fig 1a).  (Fig 1c); however, we did not observed any significant difference in TGFβR1 levels (Fig  1b).Transcript levels of SMAD4, a key downstream gene of TGFβR2, were significantly reduced in patients [median (IQR) = 0.0075 (0.005-0.009)] compared to controls [median (IQR) = 0.0087 (0.0063-0.0132)] (p = 0.043) (Fig. 2a),but no change was observed in the inhibitory SMAD7 (Fig.2b).Transcript levels of all the five genes were also analyzed between IM responsive and resistant patient groups. However, no major difference in the expression of the candidate genes was observed between the groups.

Identification of genetic variants in the candidate genes of TGFβ-SMAD signaling pathway
We sequenced exons and exon-intron boundaries of TGFβ1, TGFβR1, TGFβR2, SMAD4 and SMAD7 genes in a cohort of 20 patients and 5 healthy controls. Collectively, 52 variants were identified, and 33 variants were left in patients after filtering common variants. In these genetic variants, 14 were intronic, 11 were coding variants and 8 were in the untranslated region (UTR). Among intronic mutations, 11 were single-base substitution, 2 were deletion and 1 was insertion. However, 7 nonsynonymous, 2 synonymous and 2 deletion variants were discovered in the coding region (Fig.3a).
In TGFβ1 gene, 10 genetic variants were identified ( Table 2). Most of the variations were confined to small number of patients, but a non-synonymous variant in exon 1(c.29C>T, p.P10L.rs1800470) was present in 50% of patients examined. In TGFβR1, 5 genetic variants were identified, but an intronic variant (c.1024+24G>A, rs334354) was present in 40% of patients. Another variant (c.69A>G, rs868) observed in UTR was found identified in 4 patients. In TGFβR2, 5 genetic variants were observed, but all the variations were present in ≤3 patients ( Table 2). Seven genetic variants were discovered in SMAD4, 3 of them were intronic, 3 in UTR region and 1 variant was in exon 2. None of the variants were present in more than one patient, making it impossible to relate them to change in SMAD4 transcript levels or to associate with the disease ( Table 2). Six variants were observed in SMAD7. Among the variants, an intronic variant g.46474746C>T (rs3736242) was present in 8 out of 20 CML patients (Table 2).

DISCUSSION
CML is diagnosed by the presence of BCR-ABL gene and treated by Imatinibmesylate (TKI) in firstline setting. Alterations in BCR-ABL dependent and independent pathways are the cause of resistance to IM in CML 20. TGFβ-Smad is one of the key BCR-ABL independent pathways, which has been extensively studied in normal and abnormal hematopoiesis 21 . Alterations in this pathway have been implicated in lymphocytic 22 and myeloid leukemias 23 , but its role in CML is not well established so far. TGFβ-Smad signaling is known to increase the hyper-responsiveness of CML cells,leading to better response through BCR-ABL inhibition 24 . Although the pathway inhibits the activation of AKT, which is a downstream component of BCR-ABL pathway, it also leads to release of inhibitory sequestration of FOXO, which promotes quiescence in CML stem cells, and ultimately results in TKI resistance 25,26 . Present study attempted to explore more direct links between alterations in TGFβ-Smad signaling pathway and CML patients. TGFβ1, cytokine is a strong inhibitor of progenitor cell growth and differentiation, and its autocrine production maintains immature hematopoietic progenitor cells in quiescent state. Significant elevation was observed in TGFβ1 serum levels in CML patient group as compared to controls group. Higher levels of TGFβ1 have been observed in hematological malignancies 27 , and solid tumors 28,29 which is consistent with our findings.
International Journal of Hematology Oncology and Stem Cell Research ijhoscr.tums.ac.ir  Circulating TGFβ1 protein concentration levels were associated with mutation c.29C>T (rs1800470) in exon 1 of TGFβ1 gene 30,31 . We discovered this mutation in 50% of patients of the cohort selected for sequencing. Interestingly, elevated TGFβ1 levels were observed in 3 patients (serum levels available) harboring this mutation, but due to small sample size, the correlation between serum levels and 29C>T mutation could not be clearly demonstrated in our study. It lies in the conserved region and expected to be damaging by in silico analysis. It is speculated that Proline to Leucine (P10L) change modifies the peptide polarity, leading to change in protein transport rate 32 . We are the first to report this mutation in CML to the best of our knowledge.
A recent in-vitro study suggests that BCR-ABL expression enhance TGFβ1 levels and TGFβ signaling activity in CML cell lines 33 , which prompted us to inquire whether increased serum levels in our cohort are also leading to increased signaling activity. Evaluation of TGFβ1 receptor transcript levels showed significantly reduced TGFβR2 expression, which probably hamper tumor suppressive effect of TGFβ1 in CML patients. The finding was similar with an earlier study, where decreased TGFβR2 levels were reported in CML patients compared to healthy individuals 34 . The attempt to correlate the reduced transcript levels with genetic mutations in our cohort couldnot reveal significant observation as no mutation was present in enough number of patients to suggest such association. However, some important genetic variants were observed in TGFβR1 gene. Genetic variant, c.69A>G (rs868), present in 3' UTR of TGFβR1, was found in 20% (4/20) patients. In silico analysis of this variant shows the mutation site to be the target for miRNA Let7f/miRNA98 (Fig 3c). The Let7f/miR98 family is known to reduce TGFβR1 expression during embryogenesis and mutation in the binding region of this miRNA further reduces expression of gene 35 (Fig. 3b). Out of these 4 patients, 3 were IM resistant and showed first relapse after consuming standard dose (400mg O.D.) of imatinib mesylate for 6 years or more. The fourth patient harboring this variant completed sixth year of standard IM treatment and was a good responder till sample collection (Fig 3b). Correlation of this finding with clinico-demographic characteristics signaled towards the probable role of this variant in late relapse. Though this claim requires concrete evidence in a larger cohort, the hint is worth paying attention. Another variant, c.1024+24G>A (rs334354) in intron 6 of TGFβR1, discovered in 40% (8/20) of our patients is an established genetic marker for increased susceptibility for cancer 27,36 . Smad4 is a key component of TGFβ-Smad signaling and an important marker in colorectal cancer (CRC). Down-regulation of SMAD4 in CRC, due to increased miRNA, is responsible for its controlled expression 37 . Smad4 deficiency has been observed in malignancies of diverse origins like oral epithelial cells, keratinocytes, mammary cells, bile duct, odntoblasts 38,43 and leukemic cells of Chinese patients 44 . Our study findings also revealed significantly reduced SMAD4 levels along with low TGFBR2 levels. SMAD4 is essential for the formation of heterologous complex with SMAD2 and SMAD3 and its translocation into the nucleus for expression of target genes. Its low expression can be another potential reason for containment of this tumor suppressor pathway.

CONCLUSION
In conclusion, CML patients have elevated TGFβ1 serum levels and c.29C>T is the major genetic variant among TGFβ1 gene mutations. Lower transcript levels of TGFβR2 can be the possible reason of decreased signaling activity that abolishes the tumor suppressor effect of the increased TGFB1 levels. Though no significant change in the transcript levels of TGFBR1 was observed in patients compared to control, TGFBR1 levels were reduced in the patients with c.69A>G variant. We also reported low levels of SMAD4 in CML. Previous studies have also reported similar findings in various other types of cancer, including hematological malignancies such as acute myeloid leukemia and T-cell lymphoma 17,18 . Although our results are encouraging, but detailed research on TGFβ -SMAD signaling pathway in different CML models is required to substantiate our findings.